Streptomycin production using hens&#39; eggs



Patented June 29, 1954 STREPTOMYCIN PRODUCTION USING HENS EGGS Robert D; Muir',.Man1ius, andxA'lden B. Hatch, Syracuse;'N. Y., assignors to Bristol Labora-I tories Inc., Syracuse, N. Y., a corporation .of

New-York NoDr-awing, Application January -l'l, 1952, Serial, No. 266,990

4 Claims.- (Cl. 195-80) This invention--relates-- to= the production of streptomycin-by' -fermentation with strains :of

microorgan-ismssuch-asiStreptomyces gri'seus-and to--improvedmedia for use in such fermentation.

Itisan--objectof this- ;invention to provide medi'w fon the fermentationof--*Streptomyc'esgriseuswhich result in higher yieldsof streptomycin than are *achieved usingpreviously known media. 2

The production and properties of streptomycin havwbeen described: 'Schatz; Bugie- &" Waksman,

.PIOcrSOcr'E'XptI. Biol: Med, 55'; 66-9- (1944-).

We have now discovered; inaccordance-with the present invention, a process for the-produotiom of streptomycin which 'comprises growing Streptomyces .griseus under submerged aerobic conditionsima nutrient-medium containin hen s eggszaszariactorpromoting growtlt and stimulat ingsthesproduction of streptomycin, and isolating the:streptomycimthus=.producedi l Peak assay, Units/1111.v

Gonstituent(s) Percent I Runl ,Run 2 Peptone 0. 6 10 320 Egg white 1.0 1 10 I 2.0 5: I 33 Eggyolk; 1. 0 245 188' WholeEgg:- 2.0- 295 207- I 0.5' 10 144 Peptone0.6'%'p1us eggwvhite 1.0 9. 137

egg yolk; V 1.0; 667 675 2.0 174. v 270: 1.0 442' 874 whole eggsi; 2.0 486' 4831 Assays ofithe'streptomycinicontents of the=solu tionszwere carried; out? by: the. disc method-usin Waksmanfs. stram; of: 'Bs sub tilisl a :the-* test organismrr Example I A suitable aqueous medium vfor the fermentation- 01* Streptomyqes griseus No.5 5. to produce streptomycimhas the fOllOWiIlg composition:

Laboratory shake flask experiments were carriedtoutrin: which eggyolks; ..egg whites, or whole eggsawere:addecitoorsubstituted forthe'peptone.

Theieg szwere freshihen seggs'and were whipped priomtoiusezin. a mixing device such asthat' disclosed in U'.' SfPatent 2109,5011- THE resultsor't duplicate experiments were as follows:

The use-of peptone was accOmpanied by unexr plainedivariations in yields. Possibly these-were due .to. the presence orabsence oftrace elements. It, was obvious, however;- that egg yolks or. whole. 7 eggs have higher andzmore uniform results when used-.eit-heras a substitute. for peptone or vsi -Jan; adjuvant to. the :peptonein these small scale experiments.

Example. II

Experiments onalarger-scale were carried out-wunderithe following conditions;

Fatenz q; -s,

80 gallons medium were used in each tank.

Frozen eggs (tanners eggs) were added to the medium prior to sterilization after being thawed out and homogenized with a stirrer.

Peak Time of Percent Eggs added yield, peak yield,

units/ml. hr.

When no eggs were added the peak yield, reached after an average of 100 hours, averaged 291 units per ml. for six runs.

Example III The conditions were the same as in Example II except that 1% soy bean meal and 1% cerelose replaced the dextrose and peptone.

The peak yields as a function of added eggs were as follows:

In conjunction with this medium, it was seen that a smaller amount of egg (0.1 to 0.5%) was optional and that larger amounts were actually deleterious.

Example IV Conditions similar to those of Example II are used except that commercially-available, dried, powdered eggs are used. The peak yield of streptomycin is found to be greater than in experiments without eggs when the optimum concentration of the eggs is used. This optimum concentration lies between 0.1 and 3.0 percent.

The media used must contain a source of assimilable carbon, such as glucose, maltose, dextrose, potato starch, or hydrol which may be present in amounts such as 0.75 to 4.0%, preferably about one percent.

Certain inorganic nutrient salts must also be supplied. These may be selected from the following list, which gives in parentheses one amount which may be used in the media, but it is to be understood that not all or these salts need be present simultaneously: NaCl (1%), NaNOa (0.5%), K2HPO4 (0.1%) MgSO4-7H2O (0.1%), KCl (1%), CaCh (0.01%), N'HiCl (0.4%), CaCOs (0.25%), F6804. 71-120 (0.001%), ZnSO4 7H2O (0.000270), (NH4)2SO4 (0.25%), NazsOi (0.5%). Thus the sodium chloride content may vary up to 3% of the medium, with the optimal concentration usually falling within the range, 0.10 to 1.1%.

The proportion of hens eggs or their equivalent to be used in this process may vary within wide limits, but advantageously is about 0.1%-4.0% of the medium. The eggs may be used in addition to or as replacement for such customary materials as peptone, beef extract, fermentation solubles, corn steep liquor, dried brewers yeast, primary yeast, bean meals, peanut meal, cottonseed meal, flaxseed meal, and soybean meal.

The eggs to be used in this process may be obtained cheaply and advantageously by using the rejects from plates which open and either freeze or dry whole eggs for sale to bakeries and such consumers. The eggs may be fresh, dried or frozen whole eggs or egg yolks.

In preparing streptomycin by the improved process of the present invention, a medium of the above composition is placed in a suitable deep vessel having means for agitation and aeration of the medium, sterilized as by heating at C. for thirty minutes, and cooled to about 25 C. The sterilized medium is then inoculated with a culture of Streptomyces griseus. The concentration of the inoculum is somewhat arbitrary but the applicants like to use about 1% to about 10% by volume of the medium. The medium is incubated at about 22-35 and preferably about 28 C. under aerobic submerged conditions, i. e. constant agitation and aeration, An anti-frothing agent, for instance, a solution of a higher alcohol in an oleaginous medium may be introduced periodically in sumcient quantity to subdue frothing. The maximum streptomycin production under these conditions is usually reached in about three to four days. At this time the growth is removed by filtration. The streptomycin is recovered from the liquid medium by means now well known to the art, as, for example, by treating the filtrate of the culture broth with activated, charcoal to adsorb the streptomycin, and eluting the charcoal with a low normality aqueous alcoholic solution of acid to form an aqueous solution containing streptomycin from which streptomycin can readily be recovered by known methods. By this procedure yields of streptomycin, in terms of units present per milliliter of culture filtrate, average about 400 units per milliliter. It will, of course, be apparent that the yields in different batches may vary considerably due to differences in the streptomycin-producing properties of inocula and inherent differences in the eggs available from time to time.

Modifications may be made in carrying out the present invention without departing from the spirit and scope thereof, and we are to be limited only by the appended claims.

We claim:

1. A process for the production of streptomycin by a fermentation procedure, which comprises: growing Streptomyces griseus under aerobic submerged conditions in a nutrient medium containing hens eggs as a factor promoting growth and stimulating the production of streptomycin, and isolating the streptomycin thus produced.

2. A process for the production of streptomycinby a fermentation procedure, which comprises: growing Streptomyces grz'seus under aerobic submerged conditions in a nutrient medium containing hens egg yolks as a factor promoting growth and stimulating the production of streptomycin, and isolating the streptomycin thus produced.

3. A process for the production of streptomycin.

2,682,493 5 6 taining frozen hens eggs as a, factor promoting OTHER REFERENCES growtn and stimulating the production of strep- Levine et aL, Culture Media for Cultivation of tomycln, and lsolating the streptomycln thus Microorganism, 1930, Williams and Wilkins,

Produced 790 to 792, 800 to 801.

References Cited in the file of this patent v g ggz jg 'i g s igifig b gg fi gsa UNITED STATES PATENTS Dulaney Jour. Bact. 56, #3, Sept. 1948, pp. 305 Number Name Date 2,449,866 Waksman Sept. 21, 1943 10 Waksman, The Actmomycetas, 1950, pub. by 2,523,245 Coppock Sept. 19, 1950 Chronica Botanica 00., Waltham, Mass., pp. 195, 2,538,942 McDaniel Jan. 23, 1951 

1. A PROCESS FOR THE PRODUCTION OF STREPTOMYCIN BY A FERMENTATION PROCEDURE, WHICH COMPRISES: GROWING STREPTOMYCES GRISEUS UNDER AEROBIC SUBMERGED CONDITIONS IN A NUTRIENT MEDIUM CONTAINING HENS'' EGGS AS A FACTOR PROMOTING GROWTH AND STIMULATING THE PRODUCTION OF STREPTOMYCIN, AND ISOLATING THE STREPTOMYCIN THUS PRODUCED. 